Project Summary Quorum sensing is a bacterial cell-cell communication system that functions through the synthesis, secretion, and detection of signaling molecules called autoinducers. Quorum sensing enables bacteria to assess their cell density and coordinate group behaviors that are advantageous at high cell density. Many bacteria that cause acute and chronic infections in humans use quorum sensing to control expression of virulence factors and formation of biofilms, so inhibitors of quorum sensing hold significant promise as novel antibacterial agents. By targeting bacterial virulence as opposed to growth and survival, quorum sensing inhibitors avoid the selective pressure for drug resistance that is inherent to traditional antibiotics. The main quorum sensing system in Gram-negative bacteria is based on the synthesis and detection of acyl homoserine lactone (HSL) autoinducers. Efforts to inhibit HSL-mediated quorum sensing have focused on developing synthetic HSL analogs that act as antagonists of HSL receptors. These compounds have been shown to block virulence controlled by quorum sensing, validating quorum sensing as an antibacterial drug target. The goal of the proposed project is to develop inhibitors of HSL synthase enzymes, an alternative quorum sensing drug target that remains underexplored. Aim 1 involves the design and synthesis of small-molecule transition state analogs of the HSL synthase enzymatic reaction, an inhibitor design approach that has produced potent inhibitors of mechanistically similar enzymes. Aim 2 involves assembling robust assay platforms that will evaluate the synthetic transition state analogs as HSL synthase enzyme inhibitors and measure their ability to suppress bacterial behaviors controlled by HSL quorum sensing. The proposed project may result in potent, broad-spectrum inhibitors of HSL quorum sensing, validating HSL synthase enzymes as a novel antimicrobial drug target.